Este projecto foi apoiado pelo NIH concede DA19112 (DGW) e DA026994 (CMO). Ilustração foi fornecido por Katherine Louderback. Experimentos foram realizados no Laboratório de Vanderbilt murino Neurocomportamentais.

1. Escrever programa para ser executado sessões de testes operante utilizando variados estímulos visuais e auditivos como reforço <ol><li> Para fixa ratio (FR) sessões: fazer sessões de uma hora de duração com a luz da casa e ventilador ligado durante a sessão. Para as sessões relação progressiva, fazer sessões de duas horas de duração. Têm ambas as alavancas estendida para a duração da sessão e contrabalançar a alavanca que é designado &quot;ativa&quot; versus &quot;inativo&quot; em animais (atribuição alavanca para cada animal nunca muda). </li><li> O código do programa de tal forma que cada reforçador é variada de acordo com os seguintes parâmetros: <ul><li> cada reforçador tem uma das seguintes durações escolhidas aleatoriamente: 2, 4, 6 ou 8 seg. </li><li> cada reforçador tem uma das seguintes taxas de estímulo lâmpada de flash escolhido aleatoriamente: 0,625, 1,25, 2,5, ou 5 Hz, cada um com um ciclo de trabalho de 50%. </li><li> cada estímulo lâmpada de flash é aleatoriamente na esquerda ou no lado direito da câmara. </li><li> proporcionar um estímulo auditivo para a duração do reforçador, em nosso laboratório, provocar uma bomba de infusão que fornece cerca de 3 dB de som acima do ruído de fundo na câmara. </li></ul></li><li> O código do programa para mostrar os seguintes valores em tempo real: 1. Número de prensas alavanca ativa, 2. Número de prensas alavanca inativos, 3. Número de reforços, 4. Tempo (em incrementos de 0,1 segundo). </li></ol> 2. Lidar com animais (3 procedimento dia) <ol><li> Após aclimatação à facilidade animal, começam manejo dos animais. Isto irá habituar os animais a serem apanhados e transportados. </li><li> Comece por colocar as mãos enluvadas na gaiola e deixá-los sentar-se por 90 seg. Se cada rato não investigou as mãos naquele tempo, suavemente move as mãos para ratos e esperar que cada um deles para cheirar e / ou entre em contato com as mãos antes de prosseguir. </li><li> Com cuidado, pegue cada rato, um por um pela base da cauda e colocá-lo em sua mão, rapidamente levantando-a e trazendo-o de volta para permitir que o mouse para andar fora de sua mão de volta para a jaula. </li><li> Repita para cada rato 50-10 vezes, dependendo do comportamento do mouse. A última vez feito isso, segurar a alta do rato (com ele em pé sobre sua mão) por um período de tempo, dependendo do dia. Dia 1: 5 sec, Dia 2: 10 seg, Dia 3: 15 seg. Garantir que cada rato atende a esse critério para o dia. No dia 1, trocam as luvas entre gaiolas. </li><li> Início no dia 2, de volta curso do animal enquanto o mouse está em sua mão. Também no dia 2, começam com peso diário dos animais. Mark cauda de cada animal com um Sharpie para indicar número de registro assunto. </li><li> Se as injeções serão dadas durante o experimento, os ratos começam habituando às injeções no dia 2. Isto deve ser feito após todos os ratos na gaiola ter sido manipulados e preencheram o critério para o dia. </li></ol> 3. Equipamento limpo e teste <ol><li> Lavar panela de fundo com água quente. </li><li> Limpar paredes da câmara operante e pisos com etanol 30%. </li><li> Executar um programa de teste que liga farol, ventilador, e as luzes de estímulo, se estende alavancas, e pressiona os registros alavanca. </li><li> Assegurar que todas as luzes e os ventiladores estão funcionando corretamente e testar o programa para garantir que todas as prensas de alavanca são gravadas. </li><li> Alavancas limpa com etanol 30%. </li></ol> 4. Realizar sessão operante (sessões devem ser 5-6 dias / semana, ao mesmo tempo do dia) <ol><li> Pesar cada rato. </li><li> Carregar o programa em cada câmara e anotar o experimento de forma adequada. A alavanca ativa deve ser contrabalançada entre os animais (ou seja, a alavanca ativa é atribuída a alavanca esquerda para a metade dos ratos; a alavanca direita está ativo para a outra metade), mas o lado da alavanca ativa nunca muda uma vez um rato do rato foi atribuído. </li><li> Transporte de cada rato para sua câmara designado, feche a câmara e iniciar a sessão. </li><li> Após a sessão terminar, retire imediatamente o mouse e re-marca da cauda. </li><li> Câmaras limpas como descrito na Seção 3. </li><li> Analisar os dados para o número de prensas alavanca ativos e inativos. Número de reforços e / ou precisão alavanca (prensas% ativo alavanca) também podem ser relatados. Se os ratos vão ser testadas para o efeito de um tratamento no OSS, assegurar que todos os ratos para ser testado se encontraram critérios de aquisição (por exemplo,&gt; 20 ativos prensas alavanca e&gt; 65% prensas alavanca ativa para FR-1 final de três sessões ) antes do início do tratamento. </li><li> Após a aquisição do FR-1 de responder, o esquema de reforçamento pode ser alterado (por exemplo, uma maior proporção fixa, razão progressiva, razão aleatória, etc.) </li></ol> 5. Resultados representante Um exemplo de aquisição OSS por camundongos machos C57Bl/6J é mostrado na Figura 1 (reproduzidas a partir de 23). Camundongos controle foram submetidos condições idênticas, exceto que pressiona a alavanca de cada alavanca não tinha conseseqüência. Outro grupo de ratos é mostrado na Figura 2. Neste experimento, um grupo de ratos recebeu reforço OSS, enquanto outro grupo recebeu o reforço alimentar. Nós descobrimos que, em condições de acesso ad libitum ao alimento, tanto proporção fixa e progressiva responder são semelhantes entre OSS e respondendo por 10% Garantir (Figura 2, A e B). Isto permite comparações eficazes de uma manipulação em dois tipos diferentes de reforço (sensorial e alimentos) que evita confunde potenciais resultantes de um estado de fome ou as diferenças na taxa de resposta. <img src="/files/ftp_upload/2292/2292fig1.jpg" alt="Figura 1" /> Figura 1:. Prensas Lever por ratos OSS e controles ratos OSS recebeu variados estímulos visuais e auditivos após cada ativo alavanca de imprensa (FR-1 esquema de reforçamento), enquanto pressiona a alavanca inativo apresentou nenhuma conseqüência. Camundongos controle foram submetidos as mesmas condições, mas não houve conseqüência de tanto pressionar a alavanca (alavancas são denotados 1 e 2 e são contrabalançados em animais da mesma maneira que as alavancas ativos e inativos são contrabalançados por ratos OSS). Alavanca ativa pressionando por ratos OSS foi aumentada em relação à alavanca inativos pressionando (* p &lt;0,05, ** p &lt;0,01) e não-reforçada alavanca pressionando por controles (n = 7, 8, $ p &lt;0,05, p $ $ &lt;0,01 ). Figura reproduzida a partir de 23. <img src="/files/ftp_upload/2292/2292fig2.jpg" alt="Figura 2" /> Figura 2:. Prensas Lever por ratos e camundongos OSS responder por alimentos A.) Ratos respondeu em um cronograma de FR-1 de reforço para os estímulos OSS ou reforçador de alimentos (10% Certifique). Todos os animais tiveram acesso ad libidum aos alimentos para a duração dos experimentos. B.) Após FR-1 sessões, os camundongos foram avançadas para uma relação progressiva (PR) cronograma de reforço para cinco dias. Os dados representam as médias dos valores dos dias quatro e cinco para cada animal. A proporção final concluída é relatado à direita do eixo Y e refere-se ao número de respostas necessárias para obter o reforço correspondente (ou seja, 30 respostas são necessárias para obter o 10 º reforço após previamente ganhando nove reforços).

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Não há conflitos de interesse declarados.

Sensação operante busca é uma alternativa útil de drogas intravenosas de auto-administração, quando o rato é o animal de escolha. O fato de que nem a cirurgia nem a manutenção do cateter é necessário é vantajosa, tal como estes são importantes obstáculos técnicos no mouse. OSS também é atraente porque pode ser medida de reforço aspectos distintos de outros reforçadores naturais, como alimentos. É interessante notar que as medidas comportamentais no rato pode ser altamente...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Drug self-administration test package for mouse: extra-wide chamber and retractable levers</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>MED-307W-CT-D1</td>
<td>Levers are ultra-sensitive (require ~2 grams force) and are mounted 2.2 cm above the floor. Yellow stimulus lamps are mounted 2 cm above each lever.</td>
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<td>Interface and software package</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>MED-SYST-16</td>
<td>This is the package for up to 16 chambers.</td>
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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.

Watch this Scientific Journal Video about Operant Sensation Seeking in the Mouse at JoVE.com

Operant Sensation Seeking in the Mouse

Neste protocolo, descrevemos um método de aprendizagem operante utilizando estímulos sensoriais como reforço no mouse. Não requer treinamento prévio ou restrição alimentar, e permite o estudo do comportamento motivado, sem o uso de um reforçador farmacológicos ou naturais, tais como alimentos.

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 ...

Research

JoVE Journal

Neuroscience

12.9K visualizações.  Vanderbilt University Medical Center. Neste protocolo, descrevemos um método de aprendizagem operante utilizando estímulos sensoriais como reforço no mouse. Não requer treinamento prévio ou restrição alimentar, e permite o estudo do comportamento motivado, sem o uso de um reforçador farmacológicos ou naturais, tais como alimentos.

Vídeo: Operant Sensation Seeking in the Mouse

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

Um Procedimento para o Estudo do Reintegração Footshock Induzida de cocaína Buscando em ratos de laboratório

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Neurociência

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Detecção de placas neuríticas no rato da doença de Alzheimer Modelo

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

O Mouse Teste de Natação Forçada

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

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

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

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.  ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Uma maneira simples para medir alterações na Recompensa comportamento de busca Usando<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Operante protocolos para avaliar o custo-benefício durante a tomada de decisão reforçada por roedores

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imagem de cálcio em rato de azeitona inferior

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.