Diese Arbeit wurde durch Zuschüsse aus NIH / NIAAA (AA11852 und AA007471) unterstützt.

<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>

Dieser Artikel wurde von Med Associates, Inc. finanziert

Anwendungen und Grenzen Drug Selbstverwaltung wird in Nagetieren zu modellieren Drogenabhängigkeit eingesetzt. Viele Arzneimittel des Missbrauchs, die auf diese Weise modelliert werden kann intravenös verabreicht werden, in denen der Arzneistoff direkt mit der zentralen Kammer geliefert. Dies ermöglicht eine engmaschige Überwachung der Dosis über einen Selbst-verwaltung Sitzung. Da Ethanol ist in der Regel mündlich selbst verwaltete, ist es viel schwieriger, Wirkstoffspiegel aufgrund individueller Unterschiede in der Absorption und Stoffwechsel überwachen. Durch die Verwendung von Mikrodialyse zur Probe aus dem Gehirn region of interest, sind wir nicht nur in der Lage, um das Muster von Ethanol erreicht die Region zu überwachen, aber wir sind auch in der Lage, gleichzeitig überwachen Neurotransmitter Veränderungen in der gleichen Region im Laufe der Zeit bei jedem Selbstverwaltung Phase. Neurochemischen Änderungen und Medikamenten-induzierten Reaktionen im Gehirn mit Drogenmissbrauch und Abhängigkeit verbunden, so die Fähigkeit, gleichzeitig measicher, neurochemische und Wirkstoffkonzentrationen während bestimmter Selbstverwaltung Phasen bietet eine sehr mächtige und einzigartige Werkzeug. Ein Problem im Auge zu korrelieren Dialysat Konzentrationen an Analyten mit dem Verhalten zu halten, ist die physikalischen Eigenschaften der Mikrodialyse Sanitär. Insbesondere ist die Zeit, die für Fluid aus dem Lumen der Mikrodialysesonde dem Sammelröhrchen übertragen kritisch und kürzere Zeiten sind besser. In unserem Labor konstruieren wir die Sonden so dass die Zeit etwa 90 Sekunden. Mit diesen Bedingungen haben wir gefunden, dass es eine accumbal Dopamin Anstieg zu Beginn der Ethanol Verbrauch, der im Verlauf von der Getränkeindustrie und post-Getränk Perioden als den Dialysat Ethanolkonzentration abnimmt. 2,3,8,9 Diese Experimente , wenn sie mit Daten aus pharmakologischen Studien kombiniert, haben uns erlaubt, zu analysieren, die strikt pharmakologische Wirkungen von Ethanol und Selbstverwaltung verbunden Umweltreize auf changes in Neurotransmitter-Konzentrationen. Es sei darauf hingewiesen, dass diese spezielle Anwendung der kombinierten Verhaltens-Mikrodialyse-Techniken auf die aktuellen Forschungsinteressen unserem Labor wird geeignet sein. Es wurde entwickelt, um das zeitliche Muster von Ethanol das Gehirn erreicht im Vergleich zu dem Muster der Neurotransmitter ändert sich in der Region auszuwerten, so dass wir diese Maßnahmen auch auf synchrone Selbstverabreichung Verhaltensweisen beziehen. Die abgeleiteten Dialysat Ethanolkonzentrationen sind nicht für in vivo-Sonde Recovery korrigiert und sind nur ein Bruchteil des Gehirns Ethanol Gewebskonzentration. Wenn quantitative Mikrodialyse Ethanol erforderlich ist, sollte die Extraktion Bruchteil Ethanol daß aus dem extrazellulären Raum in die Sonde diffundiert experimentell bestimmt werden. Siehe bisherigen Veröffentlichungen aus unserem Labor für Methoden und weitere Diskussion. 10,11,12 Obwohl dieses Protokoll zeigt die Verwendung von Gas-chromatography zusammen mit Festphasenmikroextraktion von Ethanol aus dem Kopfraum des Mikrodialyse Proben könnten andere Methoden zur Analyse der Ethanolgehalt der Mikrodialyse Probe verwendet werden. Allerdings können alternative Methoden aus einigen Nachteilen leiden. Zum Beispiel können weniger empfindlichen analytischen Methoden erfordern einen größeren Probenvolumen das Probenahmen mal größer als die hier dargestellten 5-7 min erfordert. Die Art der hier diskutierten System verwendet eine Festphasenmikroextraktion, die Ethanol konzentriert in der Dampfphase in dem abgedichteten Probenphiole indem Absorption an die Faser in der Phiole Kopfraum platziert. Dies verbessert die Nachweisgrenze im Vergleich zu direkten Kopfraum Probenahme ermöglicht die typischerweise 50-100 Mikroliter des Dampfes zu injizieren. Ein weiterer wesentlicher Vorteil der Dampfraum-Verfahren ist, dass die Probe zur Analyse injiziert extrem sauber und frei von Salzen ist. Direkte Injektion der Flüssigkeit Mikrodialyse Probe kann auch mit höheren Sensit verwendet werdenduktivität, aber dies wird mehr Instrument Ausfallzeiten durch regelmäßige Wartung für die Reinigung der injizierten Salzen erforderlich erfordern. Trouble-shooting und andere Notizen <ol><li> Bevor Ihr Experiment beginnt, geben Sie sich genügend Zeit, um zu überprüfen, dass Ihre Mikrodialyse Set-ups richtig funktionieren und ein störungsfreies schießen keine Probleme. Wir schlagen vor, dass Sie eine zusätzliche Set-up, mit ACSF perfundiert, zum Umstieg bereit sind mit haben eine Fehlfunktion set-up um Zeit zu sparen, da die meisten operante Sitzungen zu einem bestimmten Zeitpunkt auftreten, jeden Tag neigen. </li><li> Sicherstellen, dass die Mikrodialysesonden über einen Zeitraum von etwa 5 min eingesetzt sind, um die Gewebeschädigung durch Scherung des Gewebes, während die Sonde dringt das Gehirn produzierte minimieren. </li><li> Bei der Übertragung der Ratte in den operante Kammer ist es hilfreich, eine zweite Person zu unterstützen, damit die Übertragungsleitung (Teil der Mikrodialyse set-up) nicht verheddert, und so, dass der operante Programm init kanniated auf Zeit. </li><li> Stellen Sie sicher, dass Sie den Autosampler Wasserbad genügend Zeit, um die richtige Temperatur zu erreichen geben. Unserem System erfordert typischerweise zwei Stunden auf 50 ° C zu erreichen Auch versichern, dass Ihre Proben sind voll, bevor die Analyse der Proben verdampft. Wir in der Regel visuell zu bestätigen, dass dies vor Beginn der Analyse von Proben aufgetreten. </li><li> Norm gute analytische Chemie Praktiken sollten befolgt werden. Diese umfassen, sind aber nicht darauf, Validierung von Individuen, Ausrüstung und Verfahren beschränkt. In kurzen, beschäftigen wir die folgenden Richtlinien: Einzelne Benutzer sollte die Möglichkeit, reproduzierbare Peakhöhe Werte für Standard-Konzentrationen und lineare Eichkurven (R2 ≥ 0,99) über mehrere Tage erstellen zu demonstrieren. So überprüfen Sie die GC-FID-System korrekt arbeitet, sollten Standards immer analysiert werden, bevor irgendwelche Dialysat Proben injiziert werden. Die Standardkurve generiert sollte eine R2 ≥ 0,99. </li><li> Die meisten Geräte verwenden wirwurde durch Varian, Inc., die von Agilent Technologies im Jahr 2010 erworben wurde gekauft. Zu diesem Zeitpunkt erhalten Bruker Scientific Instruments Varian Labor Gaschromatographie Instrumente Geschäft. Für zukünftige Einkäufe, wenden Sie sich entweder Unternehmens. </li><li> Das vorliegende Protokoll zeigt operante Verhalten mit Ratten, aber der Betrachter sollten sich bewusst sein, dass mehr Störungen des Verhaltens, mit dem kleineren Mausmodell auftreten. Ein weiteres Problem zu beachten ist, dass die Platzierung eines Mikrodialysesonde in Hirnregionen andere als die medialen präfrontalen Kortex oder Nucleus accumbens kann operante Verhalten in einem größeren Ausmaß als hier gezeigt zu stören. Es ist wichtig, genau zu prüfen zahlreichen Verhaltensparameter um festzustellen, ob der Schaden durch Aufsetzen der Sonde verursacht zu schweren Veränderungen im Verhalten. </li></ol>

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> 95% Ethanol </td><td> Aaper Alcohol and Chemical Co., Shelbyville, KY </td><td> E190, 111000190 </td></tr><tr><td> Ultra-Pure Sucrose </td><td> MP Biomedicals, LLC, Solon, OH </td><td> 821721 </td></tr><tr><td> Ultra High Purity Helium </td><td> Air Gas </td><td> HE UHP300 </td></tr><tr><td> Luft </td><td> Air Gas </td><td> AIZ300 </td></tr><tr><td> Wasserstoff </td><td> Air Gas </td><td> AIZ300 </td></tr><tr><td> GC 2 ml-Fläschchen </td><td> Agilent </td><td> # 8010-0015 </td></tr><tr><td> GC Verschlusskappen mit PTFE / Silikon Septum </td><td> Agilent </td><td> # 8010-0084 </td></tr></tbody></table> Tabelle 1. Spezifisch Reagenzien

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

Operant Selbstverwaltung Methoden werden häufig verwendet, um die Verhaltens-und pharmakologische Wirkungen vieler Medikamente von Missbrauch, einschließlich Ethanol zu studieren. Allerdings ist Ethanol in der Regel mündlich statt intravenös wie viele andere Drogen selbst verabreicht. Die Pharmakokinetik von oral verabreichten Medikamenten sind komplexer als intravenös zu verabreichende Arzneimittel. Weil das Verständnis der Beziehung zwischen den pharmakologischen und Auswirkungen auf das Verhalten von Ethanol erfordert Kenntnisse über den zeitlichen Verlauf von Ethanol Erreichen des Gehirns während und nach dem Trinken, verwenden wir in vivo Mikrodialyse und Gaschromatographie mit Flammenionisationsdetektor zum Gehirn Dialysat Ethanolkonzentrationen im Laufe der Zeit zu überwachen. Kombiniert Mikrodialyse-behavioralen Experimenten mit dem Einsatz verschiedener Techniken. In diesem Artikel, stereotaktischen Operation, Verhaltenstraining und Mikrodialyse, die geeignet ist, eine Vielzahl von Selbstverwaltung und neu getestet werden könnenpetrochemischen centered Hypothesen, werden nur einbezogen, um zu zeigen, wie sie zu den nachfolgenden Phasen der Probennahme und Dialysat Ethanol Analyse zusammenhängen. Dialysat Ethanolkonzentration Analyse mittels Gaschromatographie mit Flammen-Ionisations-Detektion, die spezifisch für Ethanol Studien ist, wird im Detail beschrieben. Daten nach diesen Methoden hergestellten zeigen die Muster von Ethanol Erreichen des Gehirns während der Selbstverwaltung Verfahren, und wenn sie mit neurochemischen Analyse der gleichen Dialysat Proben gepaart, lässt Rückschlüsse auf die pharmakologischen und Auswirkungen auf das Verhalten von Ethanol hergestellt werden.

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

Ein Verfahren, um den Zeitverlauf der Ethanolkonzentration in den Gehirnen von Ratten während operante Ethanol bestimmen Selbstverabreichung beschrieben. Gaschromatographie mit Flammenionisationsdetektion wird verwendet, um Ethanol in den Proben zu quantifizieren Dialysat, weil es die Empfindlichkeit für die kleinen Volumina erzeugt werden, die erforderlich ist.

Microdialysis Ethanol während operante Ethanol Selbstverwaltung und Ethanol Bestimmung durch Gaschromatographie

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

Research

JoVE Journal

Neuroscience

17.4K Aufrufe.  The University of Texas at Austin. Ein Verfahren, um den Zeitverlauf der Ethanolkonzentration in den Gehirnen von Ratten während operante Ethanol bestimmen Selbstverabreichung beschrieben. Gaschromatographie mit Flammenionisationsdetektion wird verwendet, um Ethanol in den Proben zu quantifizieren Dialysat, weil es die Empfindlichkeit für die kleinen Volumina erzeugt werden, die erforderlich ist.

Serie: 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.

Beurteilung der Ultraschall-Vocalizations Während Drug Selbstverwaltung in Ratten

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

Forschung

Neurowissenschaften

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.

Operante Sensation Seeking in der Maus

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.

Ein einfacher Weg zu Ethanol Empfindlichkeit in Flies Measure

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

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.

Methoden zur intravenösen Selbstverwaltung in einem Maus-Modell

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.

Identifikation der olfaktorischen Flüchtige mittels Gaschromatographie-Multi-unit Recordings (GCMR) im Insect Antennallobus

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.

Hippocampus Insulin Mikroinjektion und<em&gt; In vivo</em&gt; Microdialysis Während 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 ...

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

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

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

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

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

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

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.

Ein Test zur Messung der Wirkung von Ethanol auf die Fortbewegungsgeschwindigkeit der<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.

Maßgeschneiderte Mikrodialysesonde Entwurf

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.

Bewertung der dopaminergen Homöostase bei Mäusen durch Verwendung von High-Performance Liquid Chromatography Analyse und Synaptosomal Dopamin-Aufnahme

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