Questo progetto è stato sostenuto da NIH concede DA19112 (DGW) e DA026994 (OCM). Illustrazione è stato fornito da Katherine Louderback. Gli esperimenti sono stati eseguiti nel laboratorio Murine Vanderbilt neurocomportamentali.

1. Scrivi programma da eseguire sessioni di test operante con vari stimoli visivi e uditivi come rinforzo <ol><li> Per il rapporto fisso (FR) sessioni: rendere le sessioni di un&#39;ora in lunghezza con luce casa e ventilatore acceso durante la sessione. Per le sessioni rapporto progressivo, rendono le sessioni di due ore di lunghezza. Hanno entrambe le leve prorogato per la durata della sessione e contrappeso che è designato leva &quot;attiva&quot; contro &quot;inattivi&quot; tra animali (assegnazione leva mai per ogni animale modifiche). </li><li> Codice del programma in modo tale che ogni rinforzo varia in base ai seguenti parametri: <ul><li> ogni rinforzo ha una delle seguenti durate scelti a caso: 2, 4, 6 o 8 sec. </li><li> ogni rinforzo ha uno dei tassi di stimolo seguenti lampada flash scelto a caso: 0,625, 1,25, 2,5 o 5 Hz, ognuno con un duty cycle del 50%. </li><li> ogni stimolo lampada flash in modo casuale a sinistra oa destra della camera. </li><li> fornire uno stimolo uditivo per tutta la durata del rinforzo; nel nostro laboratorio, abbiamo innescare una pompa per infusione, che garantisce un suono di circa 3 dB il rumore di fondo nella camera. </li></ul></li><li> Codice del programma per visualizzare i seguenti valori in tempo reale: 1. Numero di pressioni della leva attivo, 2. Numero di pressioni della leva inattivi, 3. Numero di rinforzi, 4. Tempo (in incrementi di 0,1 secondi). </li></ol> 2. Trattare gli animali (3 procedura di giorno) <ol><li> Dopo acclimatazione alla struttura animale, iniziare occupano degli animali. Questo abituare gli animali ad essere raccolto e trasportato. </li><li> Inizio mettendo le mani guantate nella gabbia e lasciandoli riposare per 90 sec. Se ogni mouse non ha indagato le mani in quel momento, muovere le mani verso il mouse e attendere ciascuno di annusare e / o contattare le mani prima di procedere. </li><li> Con attenzione raccogliere ogni mouse uno per uno dalla base della coda e metterla sulla vostra mano, alzando rapidamente e riportarla verso il basso per consentire il mouse per camminare fuori della tua mano nella gabbia. </li><li> Ripetere per ogni momento del mouse 5-10, a seconda del comportamento del mouse. Il tempo finale è fatto questo, tenere alto il mouse (con in piedi sulla vostra mano) per un periodo di tempo, a seconda del giorno. Giorno 1: 5 sec, 2 ° giorno: 10 sec, 3 ° giorno: 15 sec. Garantire che ogni mouse soddisfa questo criterio per la giornata. Il giorno 1, cambiare i guanti tra gabbie. </li><li> A partire dal 2 ° giorno, il colpo dorso dell&#39;animale, mentre il mouse è sulla vostra mano. Anche il 2 ° giorno, cominciare peso gli animali ogni giorno. Segna coda ogni animale con un pennarello per indicare il numero soggetto. </li><li> Se iniezioni sarà dato durante l&#39;esperimento, i topi cominciano abituare alle iniezioni nel Day 2. Questo dovrebbe essere fatto dopo che tutti i topi in gabbia sono state manipolate e soddisfaceva il criterio per la giornata. </li></ol> 3. Pulire e verificare attrezzature <ol><li> Lavare tegame fondo con acqua calda. </li><li> Pulire le pareti della camera operante e pavimenti con il 30% di etanolo. </li><li> Eseguire un programma di test che si accende la luce casa, ventilatore, e le luci stimolo, si estende leve, e registra presse a leva. </li><li> Assicurarsi che tutte le luci e ventilatori funzionino correttamente e testare il programma per garantire che tutte le macchine a leva sono registrati. </li><li> Leve pulito con il 30% di etanolo. </li></ol> 4. Eseguire sessione operante (sessioni dovrebbero essere 5-6 giorni / settimana alla stessa ora del giorno) <ol><li> Pesare ogni mouse. </li><li> Caricare il programma in ogni camera e annotare l&#39;esperimento in modo appropriato. La leva attivo dovrebbe essere controbilanciato tra animali (ad esempio, la leva attivo è assegnato alla levetta sinistra per la metà dei topi, la leva destra è attivo per l&#39;altra metà), ma il lato leva attivo non cambia mai una volta che il mouse di un mouse è stato assegnato. </li><li> Trasporto ogni mouse alla sua sezione designata, chiudere la camera, e avviare la sessione. </li><li> Dopo la sessione termina, rimuovere immediatamente il mouse e ri-segnare la coda. </li><li> Camere pulite come descritto nella Sezione 3. </li><li> Analizzare i dati per numero di presse a leva attivi e inattivi. Numero di rinforzi e / o l&#39;accuratezza leva (presse a leva% attivo) può anche essere segnalato. Se i topi stanno per essere testati per l&#39;effetto di un trattamento su OSS, in modo che tutti i topi da testare hanno incontrato i criteri di acquisizione (ad esempio,&gt; 20 presse a leva attiva e&gt; 65% presse a leva attiva per ultime tre sessioni di FR-1 ) prima dell&#39;inizio del trattamento. </li><li> Dopo l&#39;acquisizione della FR-1 risponde, il programma di rinforzo può essere modificato (per esempio, un più alto rapporto fisso, rapporto progressivo, rapporto casuale, ecc.) </li></ol> 5. Rappresentante Risultati Un esempio di acquisizione da parte di sesso maschile OSS topi C57BL/6J è illustrato nella figura 1 (riprodotto da 23). Topi di controllo sottoposti a condizioni identiche, tranne che preme la leva su entrambi leva non ha avuto conseguenzesequenza. Un&#39;altra coorte di topi è mostrato nella Figura 2. In questo esperimento, un gruppo di topi hanno ricevuto rinforzi OSS, mentre un altro gruppo ha ricevuto rinforzi cibo. Abbiamo scoperto che in condizioni di accesso ad libitum a cibo, sia rapporto fisso e progressivo rispondere sono simili tra OSS e la risposta per il 10% Assicurare (Figura 2, A e B). Questo permette un confronto efficace di una manipolazione su due diversi tipi di rinforzo (sensoriale e alimentare) che evita confonde potenziale risultante dallo stato di fame o le differenze nel tasso di risposta. <img src="/files/ftp_upload/2292/2292fig1.jpg" alt="Figura 1" /> Figura 1:. Presse a leva da topi OSS e OSS controlli topi hanno ricevuto vari stimoli visivi e uditivi successivo attiva la leva stampa (FR-1 programma di rinforzo), mentre preme la leva inattivo avuto alcuna conseguenza. Topi di controllo ha subito le stesse condizioni ma non ci fu alcuna conseguenza o di premere la leva (leve sono denotate 1 e 2 e si contrappongono tra gli animali nello stesso modo che le leve attivi ed inattivi sono controbilanciati per i topi OSS). Leva attiva premendo dai topi OSS è stata aumentata rispetto alla leva inattivo premendo (* p &lt;0,05, ** p &lt;0,01) e non armato leva premendo da controlli (n = 7 8, $ p &lt;0,05, $ $ p &lt;0,01 ). Figura riprodotto da 23. <img src="/files/ftp_upload/2292/2292fig2.jpg" alt="Figura 2" /> Figura 2:. Presse a leva da topi OSS e topi rispondere per il cibo A.) I topi hanno risposto su una FR-1 programma di rinforzo per stimoli OSS o rinforzo alimentare (10% Assicurare). Tutti i topi avevano accesso ad libidum al cibo per tutta la durata degli esperimenti. B.) Dopo FR-1 sessioni, i topi sono stati avanzati a un rapporto progressivo (PR) programma di rinforzo per cinque giorni. Dati rappresentano il mezzo di valori da quattro giorni e cinque per ogni animale. Il rapporto finale completa è riportata sulla destra asse Y e si riferisce al numero di risposte necessarie per ottenere il rinforzo corrispondente (cioè 30 risposte devono ottenere il rinforzo 10 ° dopo la precedente guadagnare nove rinforzi).

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Nessun conflitto di interessi dichiarati.

Sensazione operante ricerca è una valida alternativa alla droga per via endovenosa di auto-somministrazione quando il mouse è l&#39;animale di scelta. Il fatto che né interventi chirurgici né manutenzione catetere è necessario è vantaggioso, in quanto questi sono significativi ostacoli tecnici nel topo. OSS è anche interessante perché si può misurare aspetti di rinforzo distinto da altri rinforzi naturali come il cibo. E &#39;utile notare che le misure comportamentali nel topo può ...

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		<th>Material Name</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

In questo protocollo si descrive un metodo di apprendimento operante mediante stimoli sensoriali come rinforzo nel topo. Non richiede alcuna formazione precedente o restrizione alimentare, e permette lo studio del comportamento motivato, senza l&#39;utilizzo di un rinforzo farmacologiche o naturali come il cibo.

Sensation operante in Cerco il mouse

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Research

JoVE Journal

Neuroscience

12.9K Views.  Vanderbilt University Medical Center. In questo protocollo si descrive un metodo di apprendimento operante mediante stimoli sensoriali come rinforzo nel topo. Non richiede alcuna formazione precedente o restrizione alimentare, e permette lo studio del comportamento motivato, senza l'utilizzo di un rinforzo farmacologiche o naturali come il cibo.

Video: 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.

Una procedura per lo studio della Footshock indotta Ripristino di cocaina Cerco nei ratti di laboratorio

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

Ricerca

Neuroscienze

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.

Multiple-topo Imaging neuroanatomici Risonanza Magnetica

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.

Rilevazione di placche neuritiche nel modello di malattia di Alzheimer mouse

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.

La forzata mouse test Swim

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.

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

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

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.

Un modo semplice per misurare alterazioni in ricompensa il comportamento di ricerca Utilizzando<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.

Protocolli dell'operatore per valutare l'analisi costi-benefici durante la risoluzione rinforzato dai roditori

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.

Transpupillare Due fotoni In vivo Imaging della retina del topo

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 Imaging del calcio nell'oliva inferiore del topo

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.