Questa ricerca è stata sostenuta da sovvenzioni NIH / NIAAA (AA11852 e AA007471).

<ol>
	<li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> J. Vis. Exp. (20), e880 (2008).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+response+in+the+nucleus+accumbens+core-shell+border+differs+from+that+in+the+core+and+shell+during+operant+ethanol+self-administration.">The dopamine response in the nucleus accumbens core-shell border differs from that in the core and shell during operant ethanol self-administration.</a> Alcohol Clin. Exp. Res. 33, 1355-1365 (2009).</li><li>Doyon, W. M., York, J. L., Diaz, L. M., Samson, H. H., Czachowski, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+activity+in+the+nucleus+accumbens+during+consummatory+phases+of+oral+ethanol+self-administration.">Dopamine activity in the nucleus accumbens during consummatory phases of oral ethanol self-administration.</a> Alcohol Clin. Exp. Res. 27, 1573-1582 (2003).</li><li>Pettit, H. O., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dose+on+cocaine+self-administration+behavior+and+dopamine+levels+in+the+nucleus+accumbens.">Effect of dose on cocaine self-administration behavior and dopamine levels in the nucleus accumbens.</a> Brain Res. 539, 94-102 (1991).</li><li>Paxinos, G., Kus, L., Ashwell, K. W. S., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of The Rat Forebrain. , (1998).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Skoog, D. A., Holler, F. J., Nieman, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Instrumental Analysis. , (1998).</li><li>Doyon, W. M., Anders, S. K., Ramachandra, V. S., Czachowski, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+operant+self-administration+of+10%+ethanol+plus+10%+sucrose+on+dopamine+and+ethanol+concentrations+in+the+nucleus+accumbens.">Effect of operant self-administration of 10% ethanol plus 10% sucrose on dopamine and ethanol concentrations in the nucleus accumbens.</a> J Neurochem. 93, 1469-1481 (2005).</li><li>Carrillo, J., Gonzales, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+exposure+to+voluntary+ethanol+self-administration+produces+adaptations+in+ethanol+consumption+and+accumbal+dopamine+signaling.">A single exposure to voluntary ethanol self-administration produces adaptations in ethanol consumption and accumbal dopamine signaling.</a> Alcohol. 45, 559-5566 (2011).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Duvauchelle, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shell+of+the+nucleus+accumbens+has+a+higher+dopamine+response+compared+with+the+core+after+non-contingent+intravenous+ethanol+administration.">The shell of the nucleus accumbens has a higher dopamine response compared with the core after non-contingent intravenous ethanol administration.</a> Neuroscience. 154, 1042-1053 (2008).</li><li>Robinson, D. L., Lara, J. A., Brunner, L. J., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Ethanol+Concentrations+in+the+Extracellular+Fluid+of+the+Rat+Brain.">Quantification of Ethanol Concentrations in the Extracellular Fluid of the Rat Brain.</a> J. Neurochem. 75, 1685-1693 (2000).</li><li>Gonzales, R. A., McNabb, J., Yim, H. J., Ripley, T., Bungay, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Microdialysis+of+Ethanol+in+Rat+Striatum.">Quantitative Microdialysis of Ethanol in Rat Striatum.</a> Alcohol Clin. Exp. Res. 22, 858-867 (1998).</li></ol>

Questo articolo è stato finanziato dal Med Associates, Inc.

Applicazioni e limitazioni Droga auto-amministrazione è utilizzato in roditori a dipendenza del modello di droga. Molti farmaci di abuso che sono modellate in questo modo può essere somministrato per via endovenosa, in cui il farmaco viene consegnato direttamente al vano centrale. Questo consente un attento monitoraggio della dose per un auto-amministrazione sessione. Dal momento che l&#39;etanolo è di solito auto-somministrato per via orale, è molto più difficile di monitorare i livelli di farmaco a causa di differenze individuali di assorbimento e il metabolismo. Utilizzando microdialisi a campione dalla regione del cervello di interesse, non sono in grado di controllare il modello di etanolo raggiungere la regione, ma siamo anche in grado di monitorare simultaneamente i cambiamenti dei neurotrasmettitori nella stessa regione nel corso del tempo durante ogni fase di auto-somministrazione. Alterazioni neurochimiche indotte da farmaci e le risposte del cervello sono associati con abuso e di dipendenza, quindi, la possibilità di concomitanza MEAneurochimico che concentrazioni del farmaco nel corso di specifiche fasi di auto-amministrazione fornisce uno strumento molto potente e unico. Una questione da tenere a mente per correlare le concentrazioni di analiti con dialisato comportamento è le caratteristiche fisiche del l&#39;impianto idraulico microdialisi. Specificamente, il tempo necessario per il fluido da trasferire dal lume della sonda microdialisi al tubo di raccolta è critica, e riducendo i tempi sono migliori. Nel nostro laboratorio, costruiamo le sonde in modo che il tempo è di circa 90 secondi. Utilizzando queste condizioni, abbiamo scoperto che vi è un aumento della dopamina accumbal all&#39;inizio del consumo di etanolo, che diminuisce nel corso dei periodi bevande e post-bevanda al crescere dialisato concentrazione di etanolo. 2,3,8,9 Questi esperimenti , in combinazione con i dati provenienti da studi farmacologici, ci hanno permesso di analizzare gli effetti strettamente farmacologici di etanolo e di auto-somministrazione associati stimoli ambientali sul canaleanges delle concentrazioni di neurotrasmettitori. Va notato che questa particolare applicazione del combinato comportamentale-microdialisi tecniche è adatto agli attuali interessi di ricerca del nostro laboratorio. È stato progettato per valutare il pattern temporale di etanolo che raggiunge il cervello in confronto al modello di cambiamenti dei neurotrasmettitori nella stessa regione, in modo da poter collegare queste misure sincroni auto-amministrazione comportamenti. I derivati ​​concentrazioni di etanolo dialisato non sono corretti per il recupero della sonda in vivo, e sono solo una frazione della concentrazione di etanolo tessuto cerebrale. Se microdialisi quantitativa di etanolo è richiesto, la frazione di estrazione di etanolo che diffonde dallo spazio extracellulare nella sonda deve essere determinato sperimentalmente. Vedere pubblicazioni precedenti del nostro laboratorio per i metodi e le ulteriori discussioni. 10,11,12 Sebbene questo protocollo illustra l&#39;uso di gas chromatography con microestrazione in fase solida di etanolo dallo spazio di testa dei campioni di microdialisi, altri metodi per l&#39;analisi del contenuto di etanolo nel campione microdialisi potrebbe essere utilizzato. Tuttavia, metodi alternativi possono soffrire di alcuni svantaggi. Ad esempio, i metodi di analisi meno sensibili possono richiedere un volume di campione più ampio che necessita di tempi di campionamento superiore al 5-7 minuti qui illustrato. Il tipo di sistema discusso qui usa un microestrazione in fase solida che concentra l&#39;etanolo nella fase vapore nella fiala campione sigillato, consentendo l&#39;assorbimento alla fibra collocato nello spazio di testa flaconcino. Questo migliora il limite di rilevazione rispetto campionamento headspace diretta che consente tipicamente 50-100 microlitri del vapore da iniettare. Un altro grande vantaggio del metodo è che spazio di testa del campione iniettato per analisi è estremamente pulita e priva di sali. Iniezione diretta del campione microdialisi liquido può essere utilizzato anche con maggiore SENsitivity, ma ciò richiederà più tempo strumento verso il basso a causa di manutenzione ordinaria necessaria per pulire i sali iniettati. Risoluzione dei problemi e altre note <ol><li> Prima che l&#39;esperimento ha inizio, concedetevi un po &#39;di tempo per verificare che i microdialisi set-up corretto funzionamento e per problemi sparare eventuali problemi. Si consiglia di avere un extra set-up, perfusi con ACSF, pronti a passare con un malfunzionamento set-up per risparmiare tempo, in quanto la maggior parte delle sessioni operante tendono a manifestarsi in un momento specifico ogni giorno. </li><li> Assicurarsi che le sonde da microdialisi sono inseriti in un periodo di circa 5 min per minimizzare il danno ai tessuti prodotti da taglio del tessuto, come la sonda penetra il cervello. </li><li> Durante il trasferimento del ratto nella camera operante è utile avere una seconda persona assistere in modo che la linea di trasferimento (parte della microdialisi set-up) non diventa aggrovigliato, e in modo che il programma può essere operante initIATED in tempo. </li><li> Assicurarsi che vi darà l&#39;abbondanza di acqua campionatore bagno di tempo per raggiungere la temperatura adeguata. Il nostro sistema richiede in genere due ore per raggiungere i 50 ° C. Inoltre, assicurarsi che i campioni siano completamente vaporizzato prima di analizzare i campioni. Noi di solito visivamente confermare che questo si è verificato prima di iniziare l&#39;analisi dei campioni. </li><li> Standard di buone pratiche di chimica analitica devono essere seguite. Questi includono, ma non sono limitati a, convalida di individui, attrezzature e procedure. In breve, si impiegano le seguenti linee guida: i singoli utenti devono dimostrare la capacità di generare riproducibili i valori di altezza di picco per le concentrazioni di standard e lineari curve standard (R2 ≥ 0,99) nell&#39;arco di più giorni. Per verificare la GC-FID sistema funziona correttamente, le norme devono essere sempre essere analizzati prima di qualsiasi campione dializzato vengono iniettati. La curva standard generata deve avere un R2 ≥ 0,99. </li><li> La maggior parte delle apparecchiature che utilizzanoè stato acquistato attraverso Varian, Inc., che è stata acquisita da Agilent Technologies nel 2010. In questo momento, Bruker Strumenti scientifici ottenuti Varian laboratorio di gascromatografia affari strumenti. Per gli acquisti futuri, consultare entrambe le società. </li><li> Il protocollo attuale mostra un comportamento operante con i ratti, ma lo spettatore deve essere consapevole che più interruzioni di comportamento è probabile che si verificano con il modello più piccolo topo. Un altro aspetto da considerare è che l&#39;inserimento di una sonda da microdialisi in altre regioni del cervello che la corteccia prefrontale mediale e nucleo accumbens può interrompere un comportamento operante in misura maggiore rispetto a quanto mostrato qui. E &#39;importante esaminare attentamente numerosi parametri comportamentali per determinare se il danno causato dal posizionamento della sonda produce gravi alterazioni nel comportamento. </li></ol>

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> 95% etanolo </td><td> AAPER alcool e Chemical Co., Shelbyville, KY </td><td> E190, 111000190 </td></tr><tr><td> Ultra-Pure Saccarosio </td><td> MP Biomedicals, LLC, Solon, OH </td><td> 821721 </td></tr><tr><td> Ultra High Purity elio </td><td> Air Gas </td><td> HE UHP300 </td></tr><tr><td> Aria </td><td> Air Gas </td><td> AIZ300 </td></tr><tr><td> Idrogeno </td><td> Air Gas </td><td> AIZ300 </td></tr><tr><td> GC 2 ml fiale </td><td> Agilent </td><td> # 8010-0015 </td></tr><tr><td> Tappi delle fiale GC con PTFE / silicone setti </td><td> Agilent </td><td> 8010-0084 # </td></tr></tbody></table> Tabella 1. Reagenti specifici

microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography

Operante auto-somministrazione metodi sono comunemente usati per studiare gli effetti comportamentali e farmacologici di molti farmaci di abuso, tra cui l&#39;etanolo. Tuttavia, l&#39;etanolo è in genere auto-somministrato per via orale, piuttosto che per via endovenosa come molte altre sostanze d&#39;abuso. La farmacocinetica dei farmaci somministrati per via orale sono più complesse di farmaci somministrati per via endovenosa. Poiché la comprensione del rapporto tra gli effetti farmacologici e comportamentali di etanolo richiede la conoscenza dell&#39;andamento nel tempo di etanolo raggiunge il cervello durante e dopo bere, usiamo microdialisi in vivo e gascromatografia con rivelatore a ionizzazione di fiamma per monitorare cerebrali concentrazioni di etanolo dialisato nel tempo. Combinati microdialisi-comportamentali esperimenti comportano l&#39;uso di tecniche diverse. In questo articolo, chirurgia stereotassica, formazione comportamentale e microdialisi, che può essere adattato per testare una moltitudine di auto-somministrazione e neupetrolchimico ipotesi centrato, sono inclusi solo per illustrare il loro rapporto con le successive fasi di raccolta dei campioni e analisi di etanolo dialisato. Dializzato concentrazione di etanolo analisi tramite gascromatografia con rivelazione a ionizzazione di fiamma, che è specifico per studi etanolo, è descritto in dettaglio. I dati prodotti da questi metodi di rivelare il disegno di etanolo che raggiunge il cervello durante la procedura di auto-amministrazione, e se abbinato con l&#39;analisi neurochimica dei campioni dialisato stessi, permette di trarre conclusioni circa gli effetti farmacologici e comportamentali di etanolo.

Watch this Scientific Journal Video about Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography at JoVE.com

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Un metodo per determinare l&#39;andamento nel tempo della concentrazione di etanolo nel cervello di ratti durante etanolo operante autosomministrazione viene descritto. Gascromatografia con rivelazione a ionizzazione di fiamma viene utilizzato per quantificare etanolo nei campioni di dialisato, perché ha la sensibilità richiesta per i piccoli volumi generati.

Microdialisi di etanolo durante la determinazione operante auto-amministrazione ed etanolo etanolo mediante gascromatografia

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

microdialysis-ethanol-during-operant-ethanol-self-administration

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JoVE Journal

Neuroscience

17.4K Views.  The University of Texas at Austin. Un metodo per determinare l'andamento nel tempo della concentrazione di etanolo nel cervello di ratti durante etanolo operante autosomministrazione viene descritto. Gascromatografia con rivelazione a ionizzazione di fiamma viene utilizzato per quantificare etanolo nei campioni di dialisato, perché ha la sensibilità richiesta per i piccoli volumi generati.

Video: Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Valutazione di vocalizzazioni ultrasoniche Durante Drug autosomministrazione in topi

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Ricerca

Neuroscienze

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 ...

A Simple Way to Measure Ethanol Sensitivity in Flies

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

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

Un modo semplice per misurare sensibilità etanolo in Flies

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

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 ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

Identificazione dei volatili olfattive utilizzando la gas cromatografia-Multi-unit Recordings (GCMR) nel lobo antennale Insetto

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Insulina Microiniezione ippocampale e<em&gt; In vivo</emMicrodialisi&gt; Durante il test di memoria spaziale

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

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

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

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

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

Un poco costoso, test comportamentale scalabile per la misurazione etanolo sedazione sensibilità e tolleranza Rapid in<em&gt; Drosophila</em

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

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un test per misurare gli effetti di etanolo sulla velocità di Locomotion<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

Su ordine Microdialisi Probe design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Valutazione dell'omeostasi dopaminergici in topi tramite l'uso di analisi di cromatografia liquida ad alte prestazioni e assorbimento della dopamina sinaptosomiali

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...