Esta pesquisa foi suportada por concessões do 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>

Este artigo foi financiada pela Med Associates, Inc.

Aplicações e limitações Auto-administração de drogas é usado em roedores para dependência de drogas modelo. Muitas drogas de abuso que são modelados deste modo podem ser administradas por via intravenosa, em que o fármaco é distribuído directamente para o compartimento central. Isso permite um acompanhamento de perto da dose durante uma sessão de auto-administração. Uma vez que o etanol é geralmente auto-administrado oralmente, é muito mais difícil de controlar os níveis da droga devido a diferenças individuais na absorção e metabolismo. Utilizando microdiálise para amostra da região do cérebro de interesse, não só são capazes de monitorar o padrão de etanol atingindo a região, mas também são capazes de monitorar simultaneamente alterações de neurotransmissores na mesma região ao longo do tempo durante cada fase de auto-administração. Alterações neuroquímicas e respostas induzidas pela droga no cérebro está associada com abuso e dependência de drogas, assim, a capacidade para medir simultaneamenteneuroquímica certeza e concentrações de drogas durante específicas de auto-administração fases fornece uma ferramenta muito poderosa e única. Uma questão a ter em mente para correlacionar as concentrações de dialisato de analitos com comportamento são as características físicas do encanamento microdiálise. Especificamente, o tempo necessário para o fluido a ser transferido a partir do lúmen da sonda de microdiálise para o tubo de recolha é crítica, e tempos mais curtos são melhores. Em nosso laboratório, nós construir as sondas de modo que o tempo é de cerca de 90 segundos. Usando estas condições, verificou-se que há um aumento da dopamina accumbal no início do consumo de etanol, que diminui ao longo dos períodos de bebida e de pós-bebe com o aumento da concentração de etanol de dialisato. 2,3,8,9 Estas experiências , quando combinado com os dados de estudos farmacológicos, permitiram-nos para analisar os efeitos farmacológicos estritamente de etanol e de auto-administração associados sinais ambientais em changes em concentrações de neurotransmissores. Deve notar-se que esta aplicação particular de combinados comportamental microdiálise técnicas é adequada para os interesses de pesquisa do nosso laboratório. Ele foi concebido para avaliar o padrão temporal de etanol atingir o cérebro, em comparação com o padrão de alterações de neurotransmissores, na mesma região, de modo que podemos relacionar estas medidas síncronos de auto-administração comportamentos. As concentrações de etanol derivados de dialisado não foram corrigidos para a recuperação in vivo da sonda, e são apenas uma fracção da concentração de tecido cerebral de etanol. Se microdiálise quantitativa de etanol é necessário, a fracção de extracção de etanol, que se difunde a partir do espaço extracelular para dentro da sonda deve ser determinado experimentalmente. Veja publicações anteriores de nosso laboratório de métodos e uma discussão mais aprofundada. 10,11,12 Embora este protocolo ilustra o uso de gás de chromatography juntamente com microextração em fase sólida de etanol a partir do espaço livre de amostras de microdiálise, outros métodos para a análise do teor de etanol na amostra de microdiálise poderia ser utilizado. Contudo, métodos alternativos podem sofrer de algumas desvantagens. Por exemplo, menos sensíveis métodos analíticos podem exigir um maior volume de amostra que requer tempos de amostragem superior a 5-7 minutos ilustrado aqui. O tipo de sistema discutido aqui utiliza uma microextração em fase sólida que se concentra o etanol na fase de vapor no tubo de amostragem selado, permitindo a absorção para a fibra colocada no espaço superior do frasco. Isto melhora o limite de detecção em comparação com a amostragem headspace directa, que normalmente permite 50-100 microlitros do vapor a ser injectado. Outra grande vantagem do método é que o espaço superior da amostra injectada para análise é extremamente limpa e livre de sais. A injecção directa da amostra de microdiálise líquido também pode ser utilizado com maior SensITivity, mas isso vai exigir mais instrumento o tempo devido à manutenção regular necessária para limpar os sais injetados. Resolução de problemas e outras notas <ol><li> Antes de sua experiência começa, dar-se muito tempo para verificar que seus microdiálise set-ups estão funcionando corretamente e na solução de problemas quaisquer problemas. Sugerimos que você tem um extra set-up, perfundidos com ACSF, pronto para mudar-se com uma avaria set-up para poupar tempo, como a maioria das sessões operantes tendem a ocorrer em um momento específico a cada dia. </li><li> Certifique-se de que as sondas de microdiálise são inseridos ao longo de um período de cerca de 5 min para minimizar o dano de tecido produzido por corte do tecido, como a sonda penetra no cérebro. </li><li> Ao transferir o rato para a câmara operante é útil ter uma segunda pessoa auxiliar de modo a que a linha de transferência (parte da microdiálise set-up) não se tornando emaranhada, e de modo a que o programa possa ser operante o initiated a tempo. </li><li> Certifique-se de que dá ao autosampler abundância banho de água de tempo para atingir a temperatura adequada. Nosso sistema geralmente requer duas horas para chegar a 50 ° C. Além disso, garantir que as amostras são completamente vaporizado antes de analisar as amostras. Nós normalmente confirmar visualmente que isso tenha ocorrido antes de iniciar a análise da amostra. </li><li> Padrão de boas práticas de química analítica deve ser seguido. Estes incluem, mas não estão limitados a, a validação dos indivíduos, o equipamento e procedimentos. Em breve, nós empregamos as seguintes diretrizes: usuários individuais devem demonstrar a capacidade de gerar reproduzíveis valores de altura de pico para as concentrações padrão e lineares curvas padrão (R2 ≥ 0,99) de vários dias. Para verificar o sistema GC-FID está funcionando corretamente, as normas devem ser sempre ser analisados ​​antes de qualquer amostra de dialisato são injetados. A curva padrão gerada deve ter um R2 ≥ 0,99. </li><li> A maior parte do equipamento que usafoi adquirido através de Varian, Inc., que foi adquirida pela Agilent Technologies em 2010. Neste momento, Instrumentos Bruker científicos obtidos laboratório Varian cromatografia gasosa negócio instrumentos. Para futuras compras, consultar ou empresa. </li><li> O presente protocolo mostra o comportamento operante com ratos, mas o espectador deve estar ciente de que mais perturbações de comportamento é provável de ocorrer com o modelo mais pequeno rato. Outra questão a ter em conta é que a colocação de uma sonda de microdiálise em outras regiões do cérebro do que o córtex pré-frontal medial ou núcleo accumbens pode perturbar o comportamento operante em um grau maior do que o mostrado aqui. É importante examinar de perto inúmeros parâmetros comportamentais para determinar se os danos causados ​​pela colocação da sonda produz alterações graves de comportamento. </li></ol>

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Etanol 95% </td><td> AAPER Álcool e Chemical Co., Shelbyville, KY </td><td> E190, 111000190 </td></tr><tr><td> Ultra-Pura Sacarose </td><td> MP Biomedicals, LLC, Solon, OH </td><td> 821721 </td></tr><tr><td> Ultra Alta Pureza Hélio </td><td> Ar Gás </td><td> HE UHP300 </td></tr><tr><td> Ar </td><td> Ar Gás </td><td> AIZ300 </td></tr><tr><td> Hidrogênio </td><td> Ar Gás </td><td> AIZ300 </td></tr><tr><td> GC 2 ml frascos </td><td> Agilent </td><td> # 8010-0015 </td></tr><tr><td> Tampas dos frascos GC com PTFE / silicone septos </td><td> Agilent </td><td> # 8010-0084 </td></tr></tbody></table> Tabela 1. Reagentes específicos

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

Operantes de auto-administração de métodos são usados ​​para estudar os efeitos comportamentais e farmacológicos de muitas drogas de abuso, incluindo etanol. No entanto, o etanol é tipicamente auto-administrados por via oral, por via intravenosa, em vez de como muitas outras drogas de abuso. A farmacocinética dos fármacos administrados por via oral são mais complexas do que as drogas por via intravenosa. Devido a compreensão da relação entre os efeitos farmacológicos e comportamentais de etanol requer um conhecimento da evolução no tempo de etanol chegar ao cérebro, durante e depois de beber, usamos microdiálise in vivo e cromatografia gasosa com detector de ionização de chama para monitorar as concentrações de etanol no cérebro de dialisado ao longo do tempo. Combinados microdiálise-comportamentais experiências envolvem a utilização de várias técnicas. Neste artigo, a cirurgia estereotáxica, a formação e comportamento de microdiálise, que pode ser adaptado para testar uma multiplicidade de auto-administração e neurochemical centrada hipóteses, estão incluídos apenas para ilustrar como eles se relacionam com as fases subseqüentes de coleta e análise de amostras de etanol dialisado. Dialisado análise da concentração de etanol através de cromatografia em fase gasosa com detecção de ionização de chama, o qual é específico para os estudos de etanol, é descrita em detalhe. Os dados produzidos por estes métodos revelam o padrão de etanol chegar ao cérebro, durante o processo de auto-administração e, quando combinadas com a análise das amostras neuroquímica dialisato mesmas, permite tirar conclusões acerca dos efeitos farmacológicos e comportamentais de etanol.

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

Um método para determinar o curso de tempo da concentração de etanol no cérebro de ratos durante etanol operante a auto-administração é descrita. Cromatografia em fase gasosa com detecção por ionização de chama é utilizado para quantificar o etanol nas amostras de dialisato, porque não tem a sensibilidade necessária para os pequenos volumes que são gerados.

Microdiálise de etanol durante a operante determinação auto-administração de etanol e etanol por cromatografia gasosa

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 visualizações.  The University of Texas at Austin. Um método para determinar o curso de tempo da concentração de etanol no cérebro de ratos durante etanol operante a auto-administração é descrita. Cromatografia em fase gasosa com detecção por ionização de chama é utilizado para quantificar o etanol nas amostras de dialisato, porque não tem a sensibilidade necessária para os pequenos volumes que são gerados.

Vídeo: 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.

Avaliação de vocalizações ultra-som durante Drogas auto-administração em ratos

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

Neurociência

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 que procura no 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.

Uma maneira simples para medir a sensibilidade de etanol no 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.

Métodos de auto-administração intravenosa em um modelo de camundongo

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.

Identificação de voláteis olfativas utilizando cromatografia gasosa-Multi-unidade Gravações (GCMR) no lobo antenal Inseto

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.

Microinjeção insulina hipocampal e<em&gt; Em vivo</em&gt; Microdiálise Durante um teste de memória espacial

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.

Um Ensaio Behavioral Barato, escalável para medição Ethanol Sedação sensibilidade e tolerância rápida em<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.

Um ensaio para medir os efeitos do etanol sobre a velocidade de 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.

Custom-made Microdiálise Probe projeto

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.

Avaliação da homeostase Dopaminergic em camundongos pelo uso de análise de cromatografia líquida de alta performance e captação da dopamina Synaptosomal

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